TYRO3, AXL, And MERTK Research Articles

AXL Receptor Tyrosine Kinase as a Therapeutic Target in Hematological Malignancies: Focus on Multiple Myeloma.

AXL belongs to the TAM (TYRO3, AXL, and MERTK) receptor family, a unique subfamily of the receptor tyrosine kinases. Their common ligand is growth arrest-specific protein 6 (GAS6). The GAS6/TAM signaling pathway regulates many important cell processes and plays an essential role in immunity, hemostasis, and erythropoiesis. In cancer, AXL overexpression and activation has been associated with cell proliferation, chemotherapy resistance, tumor angiogenesis, invasion, and metastasis; and has been correlated with a poor prognosis. In hematological malignancies, the expression and function of AXL is highly diverse, not only between the different tumor types but also in the surrounding tumor microenvironment. Most research and clinical evidence has been provided for AXL inhibitors in acute myeloid leukemia. However, recent studies also revealed an important role of AXL in lymphoid leukemia, lymphoma, and multiple myeloma. In this review, we summarize the basic functions of AXL in various cell types and the role of AXL in different hematological cancers, with a focus on AXL in the dormancy of multiple myeloma. In addition, we provide an update on the most promising AXL inhibitors currently in preclinical/clinical evaluation and discuss future perspectives in this emerging field.

Association between AXL promoter methylation and lung function growth during adolescence

ABSTRACTAXL is one of the TAM (TYRO3, AXL and MERTK) receptor tyrosine kinases and may be involved in airway inflammation. Little is known about how epigenetic changes in AXL may affect lung development during adolescence. We investigated the association between AXL DNA methylation at birth and lung function growth from 10 to 18 years of age in 923 subjects from the Children’s Health Study (CHS). DNA methylation from newborn bloodspots was measured at multiple CpG loci across the regulatory regions of AXL using Pyrosequencing. Linear spline mixed-effects models were fitted to assess the association between DNA methylation and 8-year lung function growth. Findings were evaluated for replication in a separate population of 237 CHS subjects using methylation data from the Illumina HumanMethylation450 (HM450) array when possible. A 5% higher average methylation level of the AXL promoter region at birth was associated with a 48.4 ml decrease in mean FEV1 growth from 10 to 18 years of age in the primary study population (95% CI: −100.2, 3.4), and a 53.9 ml decrease in mean FEV1 growth from 11 to 15 years of age in the replication population (95% CI: −104.3, −3.5). One CpG locus in the promoter region, cg10564498, was significantly associated with decreased growth in FEV1, FVC and MMEF from 10 to 18 years of age and the negative associations were observed in a similar age range in the replication population. These findings suggest a potential association between AXL promoter methylation at birth and lower lung function growth during adolescence.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Abstract 1883: A novel Axl and Mertk dual inhibitor ONO-7475: A new therapeutic agent for the treatment of FLT3-ITD and -wild-type acute myeloid leukemia (AML) overexpressing

Abstract Introduction: The TAM (Tyro3, Axl, and Mertk) receptor tyrosine kinases (RTKs) together with their ligands Gas6 and Pros1 play diverse roles in survival signaling in cancers. Currently, the consensus indicates that therapeutic targeting of TAM RTKs will prove direct antitumor activity and may complement existing immunotherapy regimens to unleash the full power of the anticancer immune response. A number of small-molecule kinase inhibitors targeting TAM RTKs are being developed and tested in preclinical models. ONO-7475 is a highly potent and oral dual inhibitor of Axl and Mertk, currently in a phase 1 clinical study as a therapeutic agent for AML (NCT03176277). Our previous studies demonstrated that ONO-7475 induced complete tumor remission in the FLT3-internal tandem duplication (ITD)-positive AML cell line MV-4-11 mouse xenograft model (Yasuhiro et al., Blood 2014). However, the antitumor activity against FLT3-ITD AML cells was due to Axl inhibition (Ruvolo et al., Haematologica 2017) and the mechanism of Mertk regulation in AML has not been determined. To evaluate this question, we screened 28 AML-derived cell lines for FLT3-wild type (WT) and analyzed the influence of Mertk in proliferation. Methods: Twenty-eight FLT3-WT AML cell lines were treated with ONO-7475 for 72 hours, and the cell viabilities (IC50 values) were determined. Phospho-Axl (P-Axl), Axl and Mertk were determined by Western blot analysis. P-Mertk was determined by immunoprecipitation. Gene knockdown was achieved in FLT3-WT AML by transfecting with Axl, Mertk, Gas6, Pros1, or scrambled control siRNA. Results: Six out of 28 FLT3-WT cell lines responded to ONO-7475, and two cell lines, MOLM16 and MKPL1, were highly sensitive (IC50: 4.7nM and 83.5nM). Western blot analysis demonstrated that Axl, Mertk and P-Mertk were overexpressed in these ONO-7475-sensitive cells, and both are classified as megakaryoblastic leukemia (FAB-M7). SiRNA knockdown studies showed that the cell growth in MOLM16 was partially dependent on Mertk (63.5%) but totally dependent on double genes, Axl and Mertk. Furthermore, knockdown of the ligands Gas6 and Pros1 also partially inhibited the cell growth in MOLM16, respectively (46.0%, 49.2%). Conclusion: Our data suggest that Mertk may play an important role in survival signaling in FLT3-WT AML. Therefore, targeting both Axl and Mertk by ONO-7475 could be a new strategy for the treatment of FLT3-WT patients with AML in addition to FLT3-ITD. Additional work to investigate and clarify for target populations in both FLT3-ITD and -WT AML is currently under way. Citation Format: Kohei Tanaka, Cuifang Li, Tomomi Hirosaki, Hikaru Kato, Yoshinori Ishikawa, Miho Oka, Hiroshi Egawa, Ryohei Kozaki, Toshio Yoshizawa. A novel Axl and Mertk dual inhibitor ONO-7475: A new therapeutic agent for the treatment of FLT3-ITD and -wild-type acute myeloid leukemia (AML) overexpressing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1883.

Abstract B194: Identification and characterization of monoclonal antibodies targeting the Tyro3, Axl and MerTK (TAM) family of receptor tyrosine kinases

Abstract Members of the TAM family of receptor tyrosine kinases (RTKs) play important roles in tumorigenesis and homeostatic regulation of the immune system. Two closely related ligands, Protein S (PROS1) and Growth Arrest Specific 6 (GAS6), bind and activate the TAMs, with GAS6 activating all TAMs and PROS1 activating MerTK and Tyro3. TAM RTKs are aberrantly expressed in many types of cancer where they may promote survival, metastasis, and chemoresistance. These receptors are also expressed on innate immune cells, particularly macrophages and dendritic cells, where they play important roles in clearance of apoptotic cells and negative regulation of inflammation. In tumors, activation of TAM signaling cascades appears to promote an antiinflammatory and immunosuppressive microenvironment, in turn driving tumor growth. Thus, inhibition of TAM signaling pathways through selective targeting with monoclonal antibodies could result in direct antitumor effects and promote antitumor immunity, whereas activation of TAMs could have antiinflammatory and immunosuppressive effects. We have used a variety of antibody-discovery strategies to identify potent and selective antagonists and agonists of human and rodent TAMs. Robust preclinical model systems for characterizing the mechanistic and functional effects of these antibodies have been established using engineered and nonengineered cell lines and primary cells. These include models to assess TAM activation and downstream signaling in cells and effects on cytokine secretion in LPS-stimulated U937 monocyte-derived and primary macrophages and primary dendritic cells. Effects of TAM inhibition on T-cell responses in mixed lymphocyte reactions are also being evaluated, as are in vivo mouse models. Utilizing these models, we have identified anti-TAM antibodies with antagonist or agonist activity, suggesting they may have potential in modulating the activity of these pathways. Citation Format: Diego Alvarado, Laura Vitale, Michael Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Craig Polson, James R. Storey, Jenifer Widger, Laura Mills-Chen, Karuna Sundarapandiyan, Andrea Crocker, Colleen Patterson, Biwei Zhao, Russell A. Hammond, Li-Zhen He, Venky Ramakrishna, Joel Goldstein, Lawrence Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Identification and characterization of monoclonal antibodies targeting the Tyro3, Axl and MerTK (TAM) family of receptor tyrosine kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B194.

Epigenetic regulation of AXL and risk of childhood asthma symptoms

BackgroundAXL is one of the TAM (TYRO3, AXL and MERTK) receptor tyrosine kinases and may affect numerous immune-related health conditions. However, the role for AXL in asthma, including its epigenetic regulation, has not been extensively studied.MethodsWe investigated the association between AXL DNA methylation at birth and risk of childhood asthma symptoms at age 6 years. DNA methylation of multiple CpG loci across the regulatory regions of AXL was measured in newborn bloodspots using the Illumina HumanMethylation450 array on a subset of 246 children from the Children’s Health Study (CHS). Logistic regression models were fitted to assess the association between asthma symptoms and DNA methylation. Findings were evaluated for replication in a separate population of 1038 CHS subjects using Pyrosequencing on newborn bloodspot samples. AXL genotypes were extracted from genome-wide data.ResultsHigher average methylation of CpGs in the AXL gene at birth was associated with higher risk of parent-reported wheezing, and the association was stronger in girls than in boys. This relationship reflected the methylation status of the gene-body region near the 5′ end, for which a 1% higher methylation level was significantly associated with a 72% increased risk of ever having wheezed by 6 years. The association of one CpG locus, cg00360107 was replicated using Pyrosequencing. Increased AXL methylation was also associated with lower mRNA expression level of this gene in lung tissue from the Cancer Genome Atlas (TCGA) dataset. Furthermore, AXL DNA methylation was strongly linked to underlying genetic polymorphisms.ConclusionsAXL DNA methylation at birth was associated with higher risk for asthma-related symptoms in early childhood.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer.

The receptor tyrosine kinases (RTKs) TYRO3, AXL and MERTK (TAM) have well-described oncogenic functions in a number of cancers. Notwithstanding, TAM RTKs are also potent and indispensable inhibitors of inflammation. The combined deletion of Axl and Mertk in mice enhances chronic inflammation and autoimmunity, including increased inflammation in the gut and colitis-associated cancer. On the other hand, deletion of Tyro3 increases the risk of allergic responses. Therefore, the indiscriminate inhibition of these TAM RTKs could result in undesirable immunological diseases. Here we show that AXL, but not MERTK or TYRO3 expression is enhanced in late stage colorectal cancer (CRC) and AXL expression associates with a cell migration gene signature. Silencing AXL or the inhibition of AXL kinase activity significantly inhibits tumor cell migration and invasion. These results indicate that the selective inhibition of AXL alone might confer sufficient therapeutic benefit in CRC, while preserving at least some of the beneficial, anti-inflammatory effects of MERTK and TYRO3 RTKs.

The TAM receptor Tyro3 regulates myelination in the central nervous system.

Myelin is an essential component of the mammalian nervous system, facilitating rapid conduction of electrical impulses by axons, as well as providing trophic support to neurons. Within the central nervous system, the oligodendrocyte is the specialized neural cell responsible for producing myelin by a process that is thought to be regulated by both activity dependent and independent mechanisms but in incompletely understood ways. We have previously identified that the protein Gas6, a ligand for a family of tyrosine kinase receptors known as the TAM (Tyro3, Axl, and Mertk) receptors, directly increases oligodendrocyte induced myelination in vitro. Gas6 can bind to and activate all three TAM receptors, but the high level of expression of Tyro3 on oligodendrocytes makes this receptor the principal candidate for transducing the pro-myelinating effect of Gas6. In this study, we establish that in the absence of Tyro3, the pro-myelinating effect of Gas6 is lost, that developmental myelination is delayed and that the myelin produced is thinner than normal. We show that this effect is specific to the myelination process and not due to changes in the proliferation or differentiation of oligodendrocyte precursor cells. We have further demonstrated that the reduction in myelination is due to the loss of Tyro3 on oligodendrocytes, and this effect may be mediated by activation of Erk1. Collectively, our findings indicate the critical importance of Tyro3 in potentiating central nervous system myelination. GLIA 2017 GLIA 2017;65:581-591.

Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase–uncleavable AXL mutant caused an elevated chemoresistance in non–small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.—Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Identifying Functional Differences Between Protein S and Gas-6 in Pancreatic Cancer

Introduction: Procoagulants such as Factor IX and thrombin play major roles in cancer cell proliferation and migration; however, a role for anticoagulant proteins in cancer biology has not been elucidated. The anticoagulant Protein S (PS), its homologous protein Growth Arrest Specific protein-6 (GAS-6), and the receptors for these proteins, Tyro-3, Axl and Mertk (TAM), are over expressed in many cancer cells. TAM family receptors regulate functions such as cell survival, proliferation, migration, and apoptosis. The consequences of activation of each of these receptors varies, although the mechanism that leads to different outcomes is unknown. We hypothesized that the PS and GAS-6 ligands are responsible for the variations in the functions of these signaling cascades.Methods: We used qPCR to analyze the pancreatic cancer cell lines Miapaca-2 and Panc-1 for variations in the expression of GAS-6 and PS. We sequestered PS and GAS-6 with antibodies and used FACS analysis to detect effects on the cell cycle and on cell cycle regulators.Results: GAS-6 was observed to be highly expressed in proliferating Miapaca-2 cells compared with Panc-1 cells, whereas there was no significant difference in PS mRNA levels between these cell lines. For the cell line Miapaca-2, antibody sequestration of GAS-6 arrested the cell cycle in S-phase and increased p53 phosphorylation; conversely, inhibition of PS reduced p53 phosphorylation.Conclusion: Our results indicate that PS and GAS-6 act antagonistically in controlling pancreatic cancer cell proliferation, and we hypothesize that the ratio of GAS-6 to PS expression is key to this regulation. We will further confirm our hypothesis by overexpressing and knocking down PS and GAS-6 in the pancreatic cancer cell lines. DisclosuresNo relevant conflicts of interest to declare.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice

ABSTRACTSevere hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl−/− mice show more severe symptoms than do wild-type (Axl+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.

Effects of DNA methylation in tyrosine receptor kinase genes at birth on childhood respiratory health

Introduction: Tyro3, Axl and Mertk (TAM) receptor kinases are key signaling molecules in immune responses and may affect numerous immune-related health conditions. However the role for TAM in asthma, including epigenetic regulation of TAM, has not been well studied. Increased methylation in the promoter region may silence TAM genes and lead to overstimulation of the immune system. Methods: We aimed to investigate the effects of DNA methylation of multiple CpG loci across regulatory regions of TAM genes at birth on risk of childhood asthma symptoms. DNA methylation at birth was measured from newborn blood spots using the Illumina HumanMethylation450 array on a subset of 251 children from the Children’s Health Study. Asthma symptoms, medication use and other covariates were completed by parents through questionnaire when the children were 6 years old. HM450 data were preprocessed for background correction, normalization and filtering of unwanted probes. General logistic regression models were fit to analyze the association between asthma symptoms and DNA methylation levels adjusting for covariates. FDR was used to adjust for multiple testing. Results: Increased methylation in the AXL gene was consistently associated with higher risk of asthma, wheezing or whistling in the chest, and medication use for asthma. This relationship was mainly driven by the methylation status of the promoter region in an AXL alternative transcript, which was significantly associated with a 40% increase in risk of wheeze. In addition, genetic ancestry modified the associations, with larger associations observed in Hispanic Whites compared to Non-Hispanic Whites or Hispanics of Native American descent. Conclusions: How these differences affect AXL gene expression and further respiratory health are currently unknown but warrant investigation.

TYRO3, AXL, and MERTK Receptor Tyrosine Kinases: Is There Evidence of Direct Involvement in Development and Onset of Sjögren’s Syndrome?

Sjögren’s syndrome (SjS) is a chronic, progressive, systemic, human autoimmune disease in which an auto-inflammatory process within the salivary and lacrimal glands results in loss of saliva and tear production, respectively. In-depth analyses of the autoimmune process in humans and animal models of SjS substantiates one of the more important pathoaetiological pathways: an increased level of glandular apoptosis and/or cell lysis. We have hypothesised that failure in clearance of dying cells by macrophages, dendritic cells, and neighbouring tissues results in a sustained innate inflammatory response that transitions to autoimmunity. Since the intrinsic inhibition of inflammation following phagocytosis of dying cells is a function of a family of three receptor tyrosine kinases (RTKs) known as the TAM (Tyro3, Axl, and Mertk), we put forward the following hypothesis: based on published information and analysis of our public microarray data, the failure of TAM RTK signalling, specifically in activating suppressor of cytokine signalling (SOCS) 1 and SOCS3 (which are inhibitors of immune responses), may lead to autoimmunity, and specifically, to SjS-like disease.

The role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.

Acute and chronic inflammatory responses in the lung are associated with the accumulation of large quantities of immune and structural cells undergoing apoptosis, which need to be engulfed by phagocytes in a process called ‘efferocytosis’. Apoptotic cell recognition and removal from the lung is mediated predominantly by airway macrophages, though immature dendritic cells and non-professional phagocytes, such as epithelial cells and mesenchymal cells, can also display this function. Efficient clearance of apoptotic cells from the airways is essential for successful resolution of inflammation and the return to lung homeostasis. Disruption of this process leads to secondary necrosis of accumulating apoptotic cells, release of necrotic cell debris and subsequent uncontrolled inflammatory activation of the innate immune system by the released ‘damage associated molecular patterns’ (DAMPS). To control the duration of the immune response and prevent autoimmune reactions, anti-inflammatory signalling cascades are initiated in the phagocyte upon apoptotic cell uptake, mediated by a range of receptors that recognise specific phospholipids or proteins externalised on, or secreted by, the apoptotic cell. However, prolonged activation of apoptotic cell recognition receptors, such as the family of receptor tyrosine kinases Tyro3, Axl and MerTK (TAM), may delay or prevent inflammatory responses to subsequent infections. In this review, we will discuss recent advances in our understanding of the mechanism controlling apoptotic cell recognition and removal from the lung in homeostasis and during inflammation, the contribution of defective efferocytosis to chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease, asthma and cystic fibrosis, and implications of the signals triggered by apoptotic cells in the susceptibility to pulmonary microbial infections.

Mertk deficiency affects macrophage directional migration via disruption of cytoskeletal organization.

Mertk belongs to the Tyro3, Axl and Mertk (TAM) family of receptor tyrosine kinases, and plays a pivotal role in regulation of cytoskeletal rearrangement during phagocytosis. Phagocytosis by either professional or non-professional phagocytes is impaired in the Mertk deficient individual. In the present study, we further investigated the effects of Mertk mutation on peritoneal macrophage morphology, attachment, spreading and movement. Mertk-mutated macrophages exhibited decreased attachment, weak spreading, loss of spindle-like body shape and lack of clear leading and trailing edges within the first few hours of culture, as observed by environmental scanning electron microscopy. Time-lapse video photography recording showed that macrophage without Mertk conducted mainly random movement with oscillating swing around the cell body, and lost the directional migration action seen on the WT cells. Western blotting showed a decreased phosphorylation of focal adhesion kinase (FAK). Immunocytochemistry revealed that actin filaments and dynamic protein myosin II failed to concentrate in the leading edge of migrating cells. Microtubules were localized mainly in one side of mutant cell body, with no clear MTOC and associated radially-distributed microtubule bundles, which were clearly evident in the WT cells. Our results suggest that Mertk deficiency affects not only phagocytosis but also cell shape and migration, likely through a common regulatory mechanism on cytoskeletons.

TAM receptors support neural stem cell survival, proliferation and neuronal differentiation.

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

TAM receptor deficiency affects adult hippocampal neurogenesis.

The Tyro3, Axl and Mertk (TAM) subfamily of receptor protein tyrosine kinases functions in cell growth, differentiation, survival, and most recently found, in the regulation of immune responses and phagocytosis. All three receptors and their ligands, Gas6 (growth arrest-specific gene 6) and protein S, are expressed in the central nervous system (CNS). TAM receptors play pivotal roles in adult hippocampal neurogenesis. Loss of these receptors causes a comprised neurogenesis in the dentate gyrus of adult hippocampus. TAM receptors have a negative regulatory effect on microglia and peripheral antigen-presenting cells, and play a critical role in preventing overproduction of pro-inflammatory cytokines detrimental to the proliferation, differentiation, and survival of adult neuronal stem cells (NSCs). Besides, these receptors also play an intrinsic trophic function in supporting NSC survival, proliferation, and differentiation into immature neurons. All these events collectively ensure a sustained neurogenesis in adult hippocampus.

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.

Systemic Autoimmunity in TAM Triple Knockout Mice Causes Inflammatory Brain Damage and Cell Death

The Tyro3, Axl and Mertk (TAM) triply knockout (TKO) mice exhibit systemic autoimmune diseases, with characteristics of increased proinflammatory cytokine production, autoantibody deposition and autoreactive lymphocyte infiltration into a variety of tissues. Here we show that TKO mice produce high level of serum TNF-α and specific autoantibodies deposited onto brain blood vessels. The brain-blood barrier (BBB) in mutant brains exhibited increased permeability for Evans blue and fluorescent-dextran, suggesting a breakdown of the BBB in the mutant brains. Impaired BBB integrity facilitated autoreactive T cells infiltrating into all regions of the mutant brains. Brain autoimmune disorder caused accumulation of the ubiquitin-reactive aggregates in the mutant hippocampus, and early formation of autofluorescent lipofuscins in the neurons throughout the entire brains. Chronic neuroinflammation caused damage of the hippocampal mossy fibers and neuronal apoptotic death. This study shows that chronic systemic inflammation and autoimmune disorders in the TKO mice cause neuronal damage and death.